Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras

Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6–phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.

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Supplementary Text Figs. S1 to 19
Tables S1 to S3 Other Supplementary Material for this manuscript includes the following: MDAR Reproducibility Checklist Data S1 to S8 Fig. S1.Synthesis of M6Pn peptides via solid phase peptide synthesis (SPPS).Synthetic scheme for M6Pn2 and M6Pn5 peptides.

Chemical Synthesis Procedures
Synthesis of M6Pn-3
Deprotection of M6Pn2 and M6Pn5 peptide following TFA cleavage The cleaved peptide was dissolved in MeCN (3 ml, 0.03M) under N2.Then TMSBr (0.3 ml, 2.25 mmol, 5 eq per phosphonate; 25 eq total for 5 M6Pn) was added dropwise.The N2 needle was removed immediately and the septum was sealed with parafilm (TMSBr corrodes the needle into the reaction mixture if needle is not removed).The reaction was allowed to stir for 48 hours at room temperature and the reaction mixture was concentrated under high vacuum then freeze-dried with benzene.The dried product was dissolved in MeOH (5 ml, 8 mM) and 0.5 M sodium methoxide in methanol was added dropwise until the pH reached 9-10.The reaction was allowed to stir under N2 for 24 hours.Upon completion, the reaction was quenched with formic acid, concentrated in vacuo, and purified by C18 column (elutes between 100%-90% H2O in MeCN + 0.1% TFA) to produce a white foam (82% yield from resin).M6Pn2 or M6Pn5 peptide (1 eq) was dissolved in aqueous sodium bicarbonate (pH 8.5, 8 mM).BCN-NHS (6 eq) dissolved in DMSO was added to the peptide solution.DMSO was added (to ~30 mM) until the reaction mixture became clear, and the reaction was allowed to stir for 24 hours.Upon completion, the product was lyophilized and purified by C18 column.

M6Pn2-BCN
Fig. S3.Characterization of antibody-glycopeptide LYTACs.(A) Raw mean fluorescence intensity (MFI) for EGFR degradation in Fig. 1C.(B) Raw MFI for CA9 degradation in Fig. 1D.(C) Dose-dependent degradation of EGFR in UMRC2 cells following 48 h treatment with Ctx, Ctx-M6Pn2, or Ctx-M6Pn5 as determined by live cell flow cytometry.(D) Immunoblot analysis of EGFR degradation in UMRC2 cells treated with 10 nM Ctx or Ctx-M6Pn5 for 48h.(E) Timecourse degradation of EGFR in UMRC2 cells (left) or HeLa cells (right) as determined by

Fig. S4 .
Fig. S4.Simultaneous degradation of EGFR and c-Met.Depletion of cell surface c-Met and EGFR in HEP3B cells as determined by live-cell flow cytometry following 48 h of single treatment with 10 nM of Ctx or Ona-M6Pn or co-treatment of Ctx and Ona-M6Pn.Data represent three independent experiments, and data are shown as mean ± S.E.M. P values were determined by unpaired two-tailed t-tests.

Fig. S8 .
Fig. S8.Retromer complex genes are negative hits from the CRISPR screen.(A) CasTLE score and negative effect size of retromer complex genes.(B) Immunoblot analysis of knockout of VPS26A and SNX3.(C) UMRC2 knockout versus wild-type chromatograms.DNA sequences targeted by gRNAs are underlined.
Fig. S12.Knockout of CUL3-neddylation genes impair LYTAC-mediated degradation.(A) Quantification of immunoblot of EGFR levels in WT, CAND1, and UBA3 KO cells after treatment with 10 nM Ctx or Ctx-M6Pn for 48h.(B) Quantification of immunoblot of c-MET levels in WT and CUL3 KO cells after treatment with Ona or Ona-M6Pn for 48h.Data represent three independent experiments, and data are shown as mean ± S.E.M. P values were determined by unpaired two-tailed t-tests.NS, not significant.
Fig. S13.Cycling of neddylation is essential for LYTAC activity.(A) Immunoblot analysis of EGFR levels in UMRC2 cells pre-treated with DMSO or MLN4924 (2 µM) for 24 hours, then treated with 10 nM Ctx or Ctx-M6Pn for 24 h.(B) Degradation of cell-surface EGFR in HeLa cells pre-treated with DMSO or MLN4924 (2 µM) for 24 hours, then treated with 10 nM Ctx or Ctx-M6Pn for 24 h or 50 nM EGF for 1 h as determined by live-cell flow cytometry.(C) Degradation of cell-surface c-Met in HeLa cells pre-treated with DMSO or MLN4924 (2 µM) for 24 hours, then treated with 10 nM Ona or Ona-M6Pn for 24 h as determined by live-cell flow cytometry.

Fig. S15 .
Fig. S15.Internalization of EGFR targeting LYTAC in WT and CUL3 KO cells.Visualization of WT or CUL3 KO cells following 1.5 h incubation at 37°C with 50 nM human IgG-647 and 25 nM Ctx or Ctx-M6Pn.Images are representative of two independent experiments.Scale bar, 10 µm.

Fig. S19 .
Fig. S19.Genes involved in M6P biosynthesis pathway.(A) CasTLE score and minimum negative effect size of genes involved in M6P biosynthesis pathway.(B) Immunoblot analysis of ALG12 knockouts in UMRC2 cells.(C) UMRC2 knockout versus wild-type chromatograms.DNA sequences targeted by gRNAs are underlined.(D) Lysotracker staining in WT, ALG12 KO, and GNPTAB KO cells by confocal microscopy.(E) Immunoblot of EPDR1 and streptavidin following biotin enrichment.WT or GNPTAB KO cells were incubated in PBS (no biotin control) or 1 mg/ml solution of NHS-sulfo-biotin in PBS with gentle rocking for 30 minutes at 4°C.Lysates were harvested and enriched with streptavidin beads.